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The Hypoxia Inducible Factor - research on stimulating this

This blog post is another work in progress. In it I intend to consider the research on HIF and what the possible models are that lead from varying partial pressures of Oxygen in breathed gas (and the time of exposure) and the various possible outcomes (HIF, NRF2 and NF-κB). I shall aim to write this for people who have some scientific knowledge with links to the research. If you are reading this and would like me to expand on an explanation please comment.

Disclaimer: This is not medical advice. Before you decide to do anything get advice from your doctors. Different people are affected in different ways and expert advice on your own personal circumstances is needed.
Glucose and Oxygen
Cells in animals generate energy as a molecule called ATP (Adenosine Tri Phosphate). This can be done in a number of ways. Generally the process starts with Glucose (ignoring for now when the body is using Ketones). If there is enough Oxygen then a process called Oxidative phosphorylation occurs where the mitochondria (a chemical factory in the cell) generate energy from the reactions involved. This involves things like a chemical cycle called the Citric Acid Cycle, Krebs Cycle or Tricarboxylic Acid Cycle. Where there is not enough Oxygen cells can use Anaerobic glycolysis. This produces a lot less energy than Oxidative phosphorylation (2 ATP molecules per Glucose rather than 38). You will be used to this happening when your muscles tire out. That is because the cells are switching to Anaerobic Glycolysis which ends up with Lactic Acid. Cells also have systems to check how much Oxygen there is and this enables a cell to use Aerobic Glycolysis This generates fewer molecules of ATP than Oxidative phosphorylation (4 as opposed to 38), but can run a lot faster to replenish stores of ATP. Cancer cells often grow when running in Aerobic Glycolysis mode.

The Hypoxia Inducible Factor
HIF is a protein which switches on the genes relating to a shortage of Oxygen. HIF, however, does not only switch a cell into low oxygen (hypoxic mode), it also switches on a lot of other genes and inhibits things like mTOR. mTOR (mechanistic or mamallian Target of Rapamycin) is an enzyme that handles cell growth.

To cut to the chase HIF implies that the cell thinks things are bad, we are short of oxygen and the cell should start fixing things and behaving as if there is some external threat.

Having the cell doing housework is a good thing from time to time as the cell then functions better as broken bits are eaten up by the cell (autophagy and mitophagy).

Stem Cells and HIF
Stem Cells are the cells that are used by the body to replace defunct cells. Many diseases are caused by the body's store of stem cells being reduced and also stem cells failing to turn into the correct type of cell. HIF can help with this "Hyperbaric Oxygen Increases Stem Cell Proliferation, Angiogenesis and Wound-Healing Ability of WJ-MSCs in Diabetic Mice" (The paper linked to links to papers with human cases as well). Hyperbaric means pressure greater than normal. Now it may sound a bit odd that I am talking about Hypoxia (low Oxygen), but in fact the paper refers to Hyperoxia (high oxygen). For now lets accept that conclusion that having more health Stem Cells is a good thing.

The Normobaric Oxygen Paradox
The reason why the Low Oxygen switch (HIF) is turned on by High Oxygen levels is, in fact that it is the reversion to normal oxygen levels that matters. This is called the "Normobaric Oxygen Paradox." A further subtle point is that in fact you don't have to have pressure any greather than atmospheric pressure although you do need a high concentration of Oxygen.

Oxygen Partial Pressures
The key to all of this is that it is the amount of Oxygen dissolved in the blood serum that gets inside the cell that matters. Although Haemoglobin carries a lot more oxygen in the end the levels of oxygen dissolved in the serum which is mainly water tends to be driven by the partial pressure of Oxygen in the lungs from the air that is being breathed. Hence if you double the partial pressure of Oxygen being breathed, you double the rate at which Oxygen can get to the mitochondria (although the partial pressure at the mitochondria is a lot less). You can double the partial pressure of breathed oxygen in two ways: a) double the pressure of normal air, b) have air with 42% Oxygen rather than 21% Oxygen at normal air pressure.

Switching on HIF
At this point I will assume that switching on HIF from time to time is "a good thing". Therefore, we need to know how to switch on HIF.

One way to switch on HIF is Exercise. Intensive treadmill exercise increases expression of hypoxia-inducible factor 1α and its downstream transcript targets: a potential role in neuroplasticity. Interestingly the HIF response to exercise can reduce. Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions

Another way to switch on HIF is to use Hyperbaric Oxygen Therapy. However, you can also have a high concentration of Oxygen in normal air pressure (normobaric).

How does it work
What is clear from the data is that when HIF is switched on it switches on by a reduction in Oxygen Levels. It is also clear that there are three possible types of response that occur: HIF switches on at some level (including the level of 0), NRF2 switches on at some level (Nuclear factor (erythroid-derived 2)-like 2), and/or NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) is increased (or not). It is also clear that the time of the higher level of Oxygen and the actual partial pressure of Oxygen affect the outcome. I would hypothesise that this is because the cells have a reserve of anti-oxidants to deal with the byproducts of problematic Oxidative Phyosphorylation (which can be as many as 2% of runs of the TCA cycle). Hence I would hypothesise that the different responses depend upon the level to which the reserve of anti-oxidants is depleted. One of the key anti-oxidants is mitochondrial melatonin and melatonin is normally created by using acetyl-coA (from Serotonin) that otherwise gooes into the TCA cycle. What I shall do, is analyse the data from different studies to compare the amount of depletion and see what the outcomes are.

Consider Minutes * % PaO2 above 21%
Normal atmospheric O2 is 21% so I will calculate an excess over normal as a percentage of atmospheric pressure times the number of minutes of exposure. I know that 30 minutes of 60% Oxygen can get a response so this will give some meaningful figures. I will call this the challenge figure.

My objective in writing this is to work out what the range of effective protocols are for increasing HIF. I have gone through a number of studies and I list them and my summary conclusions below. The difficulty for anyone using either a Hyperbaric Chamber with an Oxygen Concentrator or just an Oxygen Concentrator is that it is not obvious (hopefully not) that such a protocol has been undergone. Conclusions are, however,
  1. High Oxygen levels are toxic - this is well known
  2. An increase in Oxygen levels means that more Reactive Oxygen Species are created each minute.
  3. If there is no increase in anti-oxidants (generated per minute) after a point there will be Oxidative Stress from a high level of oxygen.
  4. Ill people in high levels of oxygen for a long period of time have a higher level of mortality.
  5. It is best to avoid being in high levels of oxygen for a long time.
  6. If you want a good response from HIF having a medium high level of oxygen for a shortish period of time gets the best sort of results.
  7. It is not clear from the studies what the minimum requirement is. Personally I would think 60% Oxygen is needed, but it is quite difficult to know exactly what oxygen level anyone is getting from an Oxygen Concentrator even with a non rebreathing mask. However, one study saw a result from 30% - which strikes me as low. I remember another study which said nothing happens under 40%, but could not find a reference to it. Most studies that say anything want at least 50-60%.
  8. Calculating a challenge figure is a good guide by taking the increase in Oxygen Levels over 21% and multiplying it by the number of minutes.
  9. 20 mins at 85% gives a challenge figure of 1,280. (that would be from a normal oxygen concentrator for 20 mins allowing for a bit of dilution of the air flow.
  10. 10 mins at 85% gives a challenge figure of 640. This might work, but no-one has tested it.
  11. The challenge figure of 540 actually gives quite a good HIF response.
  12. Higher challenge figures of 4,000-5,000 in a hyperbaric chamber do get a result if tried lots of times.
  13. Some research that looks at lowish normobaric partial pressures of O2 (under 90%) for short periods of time say 1, 2, 5, 10, 20, 30 minutes would be really informative, but we don't have that as yet.
  14. Clearly initially there is no response (at 0 minutes), then there is a growing HIF response, then the HIF response goes down and is replaced by NF-κB. It could be more complex than that, but probably isn't.

Studies Analysed

Study: Increasing Oxygen Partial Pressures Induce a Distinct Transcriptional Response in Human PBMC: A Pilot Study on the “Normobaric Oxygen Paradox”
This looked at human peripheral blood mononuclear cells for HIF. It uses three Oxygen levels 30%, 100% and 140%. It calls 30% Mild, 100% High and 140% Very High. The exposure was 60 minutes giving a challenge figure of 540, 4740 and 7,140.
HIF
540 After 30 minutes HIF goes up by about 50%, Triples HIF from background 3 hours later, that reduces gradually over 24 hours to about double.
4740 After 30 minutes HIF goes up by about 50%, then it goes down gradually.
7140 After 30 minutes HIF goes up by about 25%, then it goes up slightly more, but not really significantly.
NRF2
540 After 30 minutes NRF2 goes up by about 25%, hits approx 38% after 3 hours, and about 50% after 24 hours
4740 After 30 minutes NRF2 goes up by about 50%, then it stays about the same and then reduces.
7140 After 30 minutes NRF2 goes up by about 50%, then it goes up to 100% and then stays about the same.
NF-κB
540 After 30 minutes NF-κB goes up insignificantly, then an insignificant amount further
4740 After 30 minutes NF-κB goes up by about 150%, then a bit more and then comes down
7140 After 30 minutes NF-κB goes up by about 80%, then is almost the same and comes down.


Study: Acute regulation of skeletal muscle protein metabolism by nutrients, exercise and hypoxia
This study looks at muscle response to 12.5% Oxygen. It is an interesting thesis, but does not provide any useful data for this analysis.

Study: EFFECT OF NORMOBARIC HYPEROXIA ON LEUKEMIC CELL LINES IN VITRO
This study exposed cells (B and T lymphocytes) to 2 and 18 hours of hyperoxia. The Oxygen was 65%.
This gives challenge figures of 5280 and 47,520.
IκBα
5280 B Cells 75% immediately, 100% after 6 hours, Up 20% after 24 hours
47520 B Cells 75% immediately, 100% after 6 hours, Up 20% after 24 hours
5280 T Cells 100% immediately, 90% after 6 hours, Up 20% after 24 hours
47520 T Cells slightly up immediately, 18% after 6 hours, Up 20% after 24 hours
Sadly the uncertainty in these figures means that it could be simply constant.
NF-κB
5280 Both T and B cells - nothing significant
47520 Both T and B cells - nothing significant
HIF (HIF-1α)
5280 B cells - if anything goes down, but not necessarily significant
47520 B cells - goes down a bit further earlier then comes up, but not necessarily significant
5280 B cells - goes down a bit further earlier then comes up, but not necessarily significant
47520 B cells - if anything goes down, but not necessarily significant


Serum erythropoietin levels in healthy humans after a short period of normobaric and hyperbaric oxygen breathing: the "normobaric oxygen paradox"
This looks at erythropoietin the creation of which is caused by HIF. They had 100% for two hours and 250% Oxygen for 90 minutes. This is a challenge figure of 9480 and 20,610
9,480 A 60% increase (P < 0.001) in serum EPO was observed 36 h after normobaric oxygen.
20,610 In contrast, a 53% decrease in serum EPO was observed at 24 h after hyperbaric oxygen.


Pulsed high oxygen induces a hypoxic-like response in human umbilical endothelial cells and in humans 30 mins
This used 15% and 100% Oxygen for 30 mins. Because they are doing 15% O2 as well this is not necessarily directly comparable, but I will use the 100% figures as a challenge of 2,370.
haemoglobin
2370Goes up
They also used Human umbilical vein endothelial cells in 32% Oxygen that they consider equates to breathing 100% Oxygen (a reasonable conclusion). This was for two hours. That would give a challenge of 9480.
HIF (HIF-1α)
9480 whilst in hyperoxia HIF goes down. After 4 hours is about 20% of normal and goes up a bit more to 70% at 6 hours.


The Hyperoxic-Hypoxic Paradox
This is a nice paper that explains how helpful HIF can be. They report using 240% Oxygen with 20-30 minute sessions with breaks in between. That would be a challenge figure of 4380 or 6570. The paper is really worth reading.
HIF (HIF-1α)
4380 has an unquantified effect
6570 has an unquantified effect


Hypoxia, a multifaceted phenomenon: the example of the "normobaric oxygen paradox"
"However, in our initial results we have demonstrated that hyperbaric oxygen at 2.5 atmospheres absolute did not produce the same increase in EPO as normobaric oxygen, but on the contrary caused a suppression (hence the term “Normobaric Oxygen Paradox”)". The minimal concentration of inspired oxygen seems to lie around 50 % (Ciccarella et al. 2011), increasing the inspired oxygen fraction to 100 % giving variable and less consistent results.
The actual challenge figures are not available here, but "less is more" does seem to be the order of the day.

Oxygen breathing may be a cheaper and safer alternative to exogenous erythropoietin (EPO)
This was a single person breathing 99% O2 via a cannula for 90 mins. That won't provide 99% breathed Oxygen. I am not quite sure what it would do, but a challenge figure of 5310 would be reasonable (80%).
erythropoietin
5310 has an unquantified effect


Increasing EPO using the normobaric oxygen paradox: a 'not so simple' task
A bit more on the principle that "less is more".

The normobaric oxygen paradox: a novel way to administer oxygen as an adjuvant treatment for cancer?
This suggests using some hyperoxia to kill cancer cells.

Effects of short-term hyperoxia on erythropoietin levels and microcirculation in critically Ill patients: a prospective observational pilot study
This was 2 hours of 100% Oxygen for critically ill patients a challenge of 9,480 which may be reduced because they are ill. Mechanical ventilation was used.
erythropoietin
9480 has an unquantified variable effect on different patients

Can the normobaric oxygen paradox (NOP) increase reticulocyte count after traumatic hip surgery?
This was 30 mins at 100% Oxygen a challenge figure of 2,370. reticulocyte
2370 increased the reticulocytes (immature red blood cells takes two days to mature)

The "normobaric oxygen paradox": a new tool for the anesthetist? (pdf)
"A further study by our group has shown that normobaric oxygen, given at too high concentrations or even too often, is not as effective in increasing EPO or haemoglobin. The minimal concentration of inspired oxygen as already stated, seems to lay around 40-50%, increasing the inspired oxygen fraction to 100% indeed shows very variable and less consistent results."

Acute normobaric hyperoxia transiently attenuates plasma erythropoietin concentration in healthy males: evidence against the 'normobaric oxygen paradox' theory This was 100% Oxygen for 2 hours viz a challenge figure of 9480. They found that normal air provided a circadian response in erythropoietin, but the challenge figure produced a reduction.

Long-term intermittent hyperoxic exposures do not enhance erythropoiesis This looks to be the same as "Acute normobaric hyperoxia ... " above.

Hyperoxia stimulates an Nrf2-ARE transcriptional response via ROS-EGFR-PI3K-Akt/ERK MAP kinase signaling in pulmonary epithelial cells
This looks in detail at the increases in NRF2.

Role of NRF2 in protection against hyperoxic lung injury in mice
This looks at NRF2 precisely at the end of a long term hyperoxic exposure.

Anthocyanins protect human endothelial cells from mild hyperoxia damage through modulation of Nrf2 pathway
This uses 32% Oxygen but directly on cells for 16, 24 and 48 hours. 24 kills off about 30% of cells.

Redox activation of Nrf2 & NF-κB: a double end sword?

This is interesting in looking at the way these processes are activated, but does not inform challenge issues.

The high Nrf2 expression in human acute myeloid leukemia is driven by NF-κB and underlies its chemo-resistance
This is interesting in looking at the way these processes are activated, but does not inform challenge issues.

Hyperoxia Alters Ultrastructure and Induces Apoptosis in Leukemia Cell Lines
This incubated cells for 18 hours in 60% oxygen (perhaps equivalent to breathing 200%). This did not make the cells very happy (I would think a good thing given they are cancer cells), but not directly informing challenge issues as the challenge figure was quite high.

Oxygen: A Stimulus, Not “Only” a Drug
This is a good article to read, but although it notes the general conclusions of a U shaped curve it does not suggest anything in terms of dosing.

Free radicals and tissue damage produced by exercise
"We report a two- to three-fold increase in free radical (R•) concentrations of muscle and liver following exercise to exhaustion."

A high fraction of inspired oxygen may increase mortality in intubated trauma patients - A retrospective cohort study "One-year mortality was significantly increased when patients had received an FiO2 above 80% for 3-4 hours compared to under 2 hours (hazard ratio (95% CI) 2.7 (1.3-6.0), p= 0.011). When an FiO2 above 80% had been administered for more than 4 hours, there was a trend towards a higher mortality as well, but this was not statistically significant. There was a significant, time-dependent increase in mortality for patients who had received an FiO2 greater than or equal to 60%. There was no significant relationship observed between mortality and the duration of FiO2 than or equal to 40%." Association Between Hyperoxia, Supplemental Oxygen, and Mortality in Critically Injured Patients
"During periods of hyperoxia, the adjusted risk for mortality was higher with greater oxygen administration."

Need (more than) two to Tango: Multiple tools to adapt to changes in oxygen availability
This is an interesting paper, but does not give challenge figures.

Relationship between oxidative stress and HIF-1 alpha mRNA during sustained hypoxia in humans
This paper looks at actual hypoxia.

Endocrine and Metabolic Responses to Endurance Exercise Under Hot and Hypoxic Conditions
This paper looks at actual hypoxia.

Hyperbaric oxygen effect on MMP-9 after a vascular insult
This involves using 250% Oxygen for an hour. Challenge figure of 13,740

Erythropoietin production can be enhanced by normobaric oxygen breathing in healthy humans
This had 100% Oxygen for two hours (a challenge of 9,480) which did not produce more Erythropoietin initially, but did after 24 hours

Hyperbaric oxygen therapy increases telomere length and decreases immunosenescence in isolated blood cells: a prospective trial
This has 200% Oxygen for 20 minute periods (with 5 minute breaks). A challenge of 3,580. "HIF-1 alpha levels were increased from 10.54±3.39 to 19.71±3.39 at the 60th session (p=0.006) where 2 weeks post HBOT levels of 16.81±7.65 were not significantly different from baseline (p=0.16)." Interestingly the process was repeated and showed a greater response because of the repetition.

Nrf2 mediates redox adaptations to exercise
This is useful as a background paper on this issue.

Antioxidant responses and cellular adjustments to oxidative stress
This is useful as a background paper on this issue.

Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing
This is another useful background paper

Increase in endogenous erythropoietin synthesis through the normobaric oxygen paradox in cardiac surgery patients
This was 100% for 2 hours a challenge figure of 9,480 - there was a response

EPO modulation in a 14-days undersea scuba dive
This was unusual because of the length of exposure. EPO went down whilst the dive was on but increased after the end of the dive. It was about 40% Oxygen (in 2 ATA) for 14 days. This is in fact a challenge of 6384. Hemoglobin and Erythropoietin After Commercial Saturation Diving This was also a lot of long dives, but the lenght varied. It does have a similar outcome to the 14 day diving.

The Effects of Hyperbaric Oxygen at Different Pressures on Oxidative Stress and Antioxidant Status in Rats
This had the rats in Hyperbaric conditions for 24 hours the conclusion is "Conclusions: The present study showed that pressure and frequency of exposure are important factors to consider when investigating HBO2-induced oxidative stress and antioxidant response."

Effects of Hyperbaric Oxygen Therapy on Mitochondrial Respiration and Physical Performance in Middle-Aged Athletes: A Blinded, Randomized Controlled Trial
Conclusion: HBOT enhances physical performance in healthy middle-age master athletes, including VO2max, power and VO2AT. The mechanisms may be related to significant improvements in mitochondrial respiration and increased mitochondrial mass.

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